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Dehydration of cells by acute hyperosmotic stress has profound effects upon cell structure and function. Interphase chromatin and mitotic chromosomes collapse (“congelation”). HL-60/S4 cells remain ~100% viable for, at least, 1 hour, exhibiting shrinkage to ~2/3 their original volume, when placed in 300mM sucrose in tissue culture medium. Fixed cells were imaged by immunostaining confocal and STED microscopy. At a “global” structural level (μm), mitotic chromosomes congeal into a residual gel with apparent (phase) separations of Ki67, CTCF, SMC2, RAD21, H1 histones and HMG proteins. At an “intermediate” level (sub-μm), radial distribution analysis of STED images revealed a most probable peak DNA density separation of ~0.16 μm, essentially unchanged by hyperosmotic stress. At a “local” structural level (~1-2 nm), in vivo crosslinking revealed essentially unchanged crosslinked products between H1, HMG and inner histones. Hyperosmotic cellular stress is discussed in terms of concepts of mitotic chromosome structure and liquid-liquid phase separation.

Von Willebrand factor (VWF) is a multimeric hemostatic protein that is synthesized in endothelial cells, where it is stored for secretion in elongated secretory organelles called Weibel-Palade bodies (WPB). The hemostatic activity of VWF is strongly related to the length of these bodies, but how endothelial cells control the dimensions of their WPB is unclear. In this study, using a targeted short hairpin RNA screen, we identified longin-SNARE Sec22b as a novel determinant of WPB size and VWF trafficking. We found that Sec22b depletion resulted in loss of the typically elongated WPB morphology together with disintegration of the Golgi and dilation of rough endoplasmic reticulum cisternae. This was accompanied by reduced proteolytic processing of VWF, accumulation of VWF in the dilated rough endoplasmic reticulum and reduced basal and stimulated VWF secretion. Our data demonstrate that the elongation of WPB, and thus adhesive activity of their cargo VWF, is determined by the rate of anterograde transport between endoplasmic reticulum and Golgi, which depends on Sec22b-containing SNARE complexes.

Epigenomic regulation plays a vital role in cell differentiation. The leukemic HL-60/S4 [human myeloid leukemic cell line HL-60/S4 (ATCC CRL-3306)] promyelocytic cell can be easily differentiated from its undifferentiated promyelocyte state into neutrophil- and macrophage-like cell states. In this study, we present the underlying genome and epigenome architecture of HL-60/S4 through its differentiation. We performed whole-genome bisulphite sequencing of HL-60/S4 cells and their differentiated counterparts. With the support of karyotyping, we show that HL-60/S4 maintains a stable genome throughout differentiation. Analysis of differential Cytosine-phosphate-Guanine dinucleotide methylation reveals that most methylation changes occur in the macrophage-like state. Differential methylation of promoters was associated with immune-related terms. Key immune genes, CEBPA, GFI1, MAFB and GATA1 showed differential expression and methylation. However, we observed the strongest enrichment of methylation changes in enhancers and CTCF binding sites, implying that methylation plays a major role in large-scale transcriptional reprogramming and chromatin reorganisation during differentiation. Correlation of differential expression and distal methylation with support from chromatin capture experiments allowed us to identify putative proximal and long-range enhancers for a number of immune cell differentiation genes, including CEBPA and CCNF. Integrating expression data, we present a model of HL-60/S4 differentiation in relation to the wider scope of myeloid differentiation., Summary: Epigenomics play a role in cell identity and differentiation. We present the DNA methylation landscape of leukemic cells during in vitro differentiation, adding another omics layer to better understand differentiation mechanisms.

Epichromatin is identified by immunostaining fixed and permeabilized cells with particular bivalent anti-nucleosome antibodies (mAbs PL2-6 and 1H6). During interphase, epichromatin resides adjacent to the inner nuclear membrane; during mitosis, at the outer surface of mitotic chromosomes. By STED (stimulated emission depletion) microscopy, PL2-6 stained interphase epichromatin is ∼76 nm thick and quite uniform; mitotic epichromatin is more variable in thickness, exhibiting a "wrinkled" surface with an average thickness of ∼78 nm. Co-immunostaining with anti-Ki-67 demonstrates Ki-67 deposition between the PL2-6 "ridges" of mitotic epichromatin. Monovalent papain-derived Fab fragments of PL2-6 yield a strikingly different punctate "chromomeric" immunostaining pattern throughout interphase nuclei and along mitotic chromosome arms. Evidence from electrophoretic mobility shift assay (EMSA) and from analytical ultracentrifugation characterize the Fab/mononucleosome complex, supporting the concept that there are two binding sites per nucleosome. The peptide sequence of the Hv3 region (heavy chain variable region 3) of the PL2-6 antibody binding site strongly resembles other nucleosome acidic patch binding proteins (especially, LANA and CENPC), supporting that the nucleosome acidic patch is included within the epichromatin epitope. It is speculated that the interphase epichromatin epitope is "exposed" with favorable geometric arrangements for binding bivalent PL2-6 at the surface chromatin; whereas, the epitope is "hidden" within internal chromatin. Furthermore, it is suggested that the "exposed" nucleosome surface of mitotic epichromatin may play a role in post-mitotic nuclear envelope reformation.

To understand the chromatin changes underlying differential gene expression during induced differentiation of human leukemic HL-60/S4 cells, we conducted RNA-Seq analysis on quadruplicate cultures of undifferentiated, granulocytic- and macrophage-differentiated cell forms. More than half of mapped genes exhibited altered transcript levels in the differentiated cell forms. In general, more genes showed increased mRNA levels in the granulocytic form and in the macrophage form, than showed decreased levels. The majority of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were significantly enriched in genes that exhibited differential transcript levels after either RA or TPA treatment. Changes in transcript levels for groups of genes with characteristic protein phenotypes, such as genes encoding cytoplasmic granular proteins, nuclear envelope and cytoskeletal proteins, cell adhesion proteins, and proteins involved in the cell cycle and apoptosis illustrate the profound differences among the various cell states. In addition to the transcriptome analyses, companion karyotyping by M-FISH of undifferentiated HL-60/S4 cells revealed a plethora of chromosome alterations, compared with normal human cells. The present mRNA profiling provides important information related to nuclear shape changes (e.g., granulocyte lobulation), deformability of the nuclear envelope and linkage between the nuclear envelope and cytoskeleton during induced myeloid chromatin differentiation.

Conversion of the pathogenic sickle allele to a naturally occurring, non-pathogenic hemoglobin variant, Hb G-Makassar, represents a long-term and durable treatment strategy for sickle cell disease (SCD). Using our engineered adenine base editor, we achieved highly efficient base editing in mobilized sickle trait (HbAS) and non-mobilized homozygous sickle (HbSS) CD34 + cells that led to >70% conversion of sickle allele to Makassar allele in in vitro erythroid differentiated (IVED) cells derived from ex vivo edited CD34 + cells. At this level of editing, >70% bi-allelic Makassar editing could be achieved in HbSS IVED cells, with ~20% of cells being mono-allelically Makassar edited. These mono-allelically edited cells behaved similarly to sickle trait (HbAS) cells, when exposed to hypoxic conditions in vitro. In vivo proof of concept xenotransplantation studies demonstrated that Makassar edited HbAS CD34 + cells achieved long-term, multi-lineage hematopoietic engraftment as well as Makassar globin protein expression in human erythroid glycophorin A + cells in thebone marrow of immunocompromised mice. Although the Makassar variant is naturally occurring in human genetics and present in individuals in Southeast Asia with normal hematologic parameters in both heterozygous and homozygous states, we sought to further characterize Makassar hemoglobin and assess its biophysical and biochemical properties. Recombinant Makassar globin was co-expressed with alpha globin in E. coli and tetramers were purified to homogeneity. Recombinant tetramers were assessed for identity, purity, globin content, and heme content demonstrating comparability to hemoglobin tetramers isolated from primary sources (whole blood). Several characterization methods were employed, to assess size, molecular weight, oligomerization state, tetramer composition, and oxygen binding properties. These studies indicated Makassar globin could properly assemble into hemoglobin tetramers, displaying biochemical properties characteristic of hemoglobins. Furthermore, we assessed polymerization potential using a temperature jump method previously employed for kinetic measurements of sickle-fiber formation and found Makassar hemoglobin did not polymerize in vitro under conditions where sickle hemoglobin (HbS) readily polymerizes, consistent with behavior observed previously by others. Finally, a crystal structure of Hb G-Makassar has been determined at the 2.2 Å resolution and showed high similarity to the HbA (wildtype hemoglobin) structure with a RMSD of 0.385 Å for all the Cα atoms, which indicates that the glutamic acid to alanine (E6A) substitution in beta-hemoglobin does not seem to induce any significant conformational change in hemoglobin structures. Altogether, our biophysical and biochemical characterization shows that the Makassar variant behaves as a functional hemoglobin. By replacing the pathogenic sickle globin with a benign hemoglobin variant with normal function, our base editing approach provides a promising autologous investigational cell therapy for the treatment of SCD. Disclosures Chu: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company, Current holder of stock options in a privately-held company. Ortega: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Feliciano: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Winton: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Xu: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company, Current holder of stock options in a privately-held company. Haupt: Beam Therapeutics: Current Employment, Current holder of stock options in a privately-held company. McDonald: Beam Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Martinez: Beam Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Liquori: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Marshall: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Lam: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company, Current holder of stock options in a privately-held company. Olins: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company, Current holder of stock options in a privately-held company. Rinaldi: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company, Current holder of stock options in a privately-held company. Rehberger: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company, Current holder of stock options in a privately-held company. Lazarra: Beam Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Decker: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company, Current holder of stock options in a privately-held company. Gantzer: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company, Current holder of stock options in a privately-held company. Bohnuud: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company, Current holder of stock options in a privately-held company. Born: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company, Current holder of stock options in a privately-held company. Barrera: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company, Current holder of stock options in a privately-held company. Yan: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company, Current holder of stock options in a privately-held company. Slaymaker: Beam Therapeutics: Current Employment, Current holder of stock options in a privately-held company, Patents & Royalties. Packer: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company, Current holder of stock options in a privately-held company. Smith: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company, Current holder of stock options in a privately-held company. Zambonelli: Beam Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Lee: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company, Current holder of stock options in a privately-held company. Gaudelli: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company, Current holder of stock options in a privately-held company. Hartigan: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company, Current holder of stock options in a privately-held company. Ciaramella: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees.

Background Mammalian cells are flexible and can rapidly change shape when they contract, adhere, or migrate. The nucleus must be stiff enough to withstand cytoskeletal forces, but flexible enough to remodel as the cell changes shape. This is particularly important for cells migrating through confined spaces, where the nuclear shape must change in order to fit through a constriction. This occurs many times in the life cycle of a neutrophil, which must protect its chromatin from damage and disruption associated with migration. Here we characterized the effects of constricted migration in neutrophil-like cells. Results Total RNA sequencing identified that migration of neutrophil-like cells through 5- or 14-μm pores was associated with changes in the transcript levels of inflammation and chemotaxis-related genes when compared to unmigrated cells. Differentially expressed transcripts specific to migration with constriction were enriched for groups of genes associated with cytoskeletal remodeling. Hi-C was used to capture the genome organization in control and migrated cells. Limited switching was observed between the active (A) and inactive (B) compartments after migration. However, global depletion of short-range contacts was observed following migration with constriction compared to migration without constriction. Regions with disrupted contacts, TADs, and compartments were enriched for inactive chromatin. Conclusion Short-range genome organization is preferentially altered in inactive chromatin, possibly protecting transcriptionally active contacts from the disruptive effects of migration with constriction. This is consistent with current hypotheses implicating heterochromatin as the mechanoresponsive form of chromatin. Further investigation concerning the contribution of heterochromatin to stiffness, flexibility, and protection of nuclear function will be important for understanding cell migration in relation to human health and disease. Electronic supplementary material The online version of this article (10.1186/s12915-018-0608-2) contains supplementary material, which is available to authorized users.

Cell differentiation is associated with changes in chromatin organization and gene expression. In this study, we examine chromatin structure following differentiation of the human myeloid leukemia cell line (HL-60/S4) into granulocytes with retinoic acid (RA) or into macrophage with phorbol ester (TPA). We performed ChIP-seq of histone H3 and its modifications, analyzing changes in nucleosome occupancy, nucleosome repeat length, eu-/heterochromatin redistribution and properties of epichromatin (surface chromatin adjacent to the nuclear envelope). Nucleosome positions changed genome-wide, exhibiting a specific class of alterations involving nucleosome loss in extended (∼1kb) regions, pronounced in enhancers and promoters. Genes that lost nucleosomes at their promoters showed a tendency to be upregulated. On the other hand, nucleosome gain did not show simple effects on transcript levels. The average genome-wide nucleosome repeat length (NRL) did not change significantly with differentiation. However, we detected an approximate 10 bp NRL decrease around the haematopoietic transcription factor (TF) PU.1 and the architectural protein CTCF, suggesting an effect on NRL proximal to TF binding sites. Nucleosome occupancy changed in regions associated with active promoters in differentiated cells, compared with untreated HL-60/S4 cells. Epichromatin regions revealed an increased GC content and high nucleosome density compared with surrounding chromatin. Epichromatin showed depletion of major histone modifications and revealed enrichment with PML body-associated genes. In general, chromatin changes during HL-60/S4 differentiation appeared to be more localized to regulatory regions, compared with genome-wide changes among diverse cell types studied elsewhere.

While there are several small molecule, gene therapy, and gene editing approaches for treating sickle cell disease (SCD), these strategies do not result in the direct elimination of the causative sickle β-globin (HbS) variant itself. The reduction or complete removal of this pathologic globin variant and expression of normal β-hemoglobin (HbB) or other non-polymerizing β-globin variant may increase the likelihood of beneficial outcomes for SCD patients. Adenine base editors (ABEs) can precisely convert the mutant A-T base pair responsible for SCD to a G-C base pair, thus generating the hemoglobin variant, Hb G-Makassar, a naturally occurring β-globin variant that is not associated with human disease. Our studies have identified ABEs that can achieve highly efficient Makassar editing (>70%) of the sickle mutation in both sickle trait (HbAS) and homozygous sickle (HbSS) patient CD34+ cells with high cell viability and recovery and without perturbation of immunophenotypic hematopoietic stem and progenitor cell (HSPC) frequencies after ex vivo delivery of guide RNA and mRNA encoding the ABE. Furthermore, Makassar editing was retained throughout erythropoiesis in bulk in vitro erythroid differentiated cells (IVED) derived from edited CD34+ cells. To gain an understanding of allelic editing at a single clone resolution, we assessed editing frequencies of clones from both single cell expansion in erythroid differentiation media, as well as from single BFU-E colonies. We found that we could achieve >70% of colonies with bi-allelic Makassar editing and approximately 20% of colonies with mono-allelic Makassar editing, while Previously, conventional hemoglobin capillary electrophoresis and high-performance liquid chromatography (HPLC) were unable to distinguish between HbS and HbG-Makassar. Here, we developed an ultra-high-performance liquid chromatography (UPLC) method that resolves sickle globin (HbS) from Hb G-Makassar globin in IVED cells. The Makassar globin variant was further confirmed by liquid chromatography mass spectrometry (LC-MS). By developing this new method to resolve these two β-globin variants in edited HbSS cells, we were able to detect, in bulk IVED cultures, >80% abundance Hb G-Makassar of total β-globins, which corresponded to a concomitant reduction of HbS levels to 70% of cells that had bi-allelic Makassar editing. Moreover, in the approximately 20% of colonies that were found to be mono-allelically edited for the Makassar variant, there was a 60:40 ratio of Hb G-Makassar:HbS globin abundance in individual clones, at levels remarkably similar to the HbA(wildtype HbB):HbS levels found in HbAS individuals, with minimal observable in vitro sickling when exposed to hypoxia. Thus, with our ABEs, we were able to reduce HbS to 90% of IVED cells and found that in vitro sickling under hypoxia inversely correlated with the level of Hb G-Makassar globin variant installation and corresponding reduction in HbS levels. By converting HbS to Hb G-Makassar, our direct and precise editing strategy replaces a pathogenic β-globin with one that has been shown to have normal hematologic parameters. Coupled with autologous stem cell transplant, this next generation gene editing strategy presents a promising new modality for treating patients with SCD. Disclosures Chu: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Lam:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Packer:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Olins:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Liquori:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Marshall:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Lee:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Yan:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Decker:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Gantzer:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Haskett:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Bohnuud:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Born:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Barrera:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Slaymaker:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Gaudelli:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Hartigan:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Ciaramella:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company.

Grass biomass is comprised chiefly of secondary walls that surround fiber and xylem cells. A regulatory network of interacting transcription factors in part regulates cell wall thickening. We identified Brachypodium distachyon SECONDARY WALL ASSOCIATED MYB1 (SWAM1) as a potential regulator of secondary cell wall biosynthesis based on gene expression, phylogeny, and transgenic plant phenotypes. SWAM1 interacts with cellulose and lignin gene promoters with preferential binding to AC-rich sequence motifs commonly found in the promoters of cell wall-related genes. SWAM1 overexpression (SWAM-OE) lines had greater above-ground biomass with only a slight change in flowering time while SWAM1 dominant repressor (SWAM1-DR) plants were severely dwarfed with a striking reduction in lignin of sclerenchyma fibers and stem epidermal cell length. Cellulose, hemicellulose, and lignin genes were significantly down-regulated in SWAM1-DR plants and up-regulated in SWAM1-OE plants. There was no reduction in bioconversion yield in SWAM1-OE lines; however, it was significantly increased for SWAM1-DR samples. Phylogenetic and syntenic analyses strongly suggest that the SWAM1 clade was present in the last common ancestor between eudicots and grasses, but is not in the Brassicaceae. Collectively, these data suggest that SWAM1 is a transcriptional activator of secondary cell wall thickening and biomass accumulation in B. distachyon.

Cycling eukaryotic cells rapidly re-establish the nuclear envelope and internal architecture following mitosis. Studies with a specific anti-nucleosome antibody recently demonstrated that the surface ("epichromatin") of interphase and mitotic chromatin possesses a unique and conserved conformation, suggesting a role in postmitotic nuclear reformation. Here we present evidence showing that the anionic glycerophospholipid phosphatidylserine is specifically located in epichromatin throughout the cell cycle and is associated with nucleosome core histones. This suggests that chromatin bound phosphatidylserine may function as a nucleation site for the binding of ER and re-establishment of the nuclear envelope.

Objective Lamin B receptor (LBR) is an integral protein of the inner nuclear membrane. Recent studies have demonstrated that genetic deficiency of LBR during granulopoiesis results in hypolobulation of the mature neutrophil nucleus, as observed in human Pelger-Huet anomaly and mouse ichthyosis ( ic ). In this study, we utilized differentiated early promyelocytes (EPRO cells) that were derived from the bone marrow of homozygous and heterozygous ichthyosis mice to examine changes to the expression of nuclear envelope proteins and heterochromatin structure that result from deficient LBR expression. Materials and Methods Wild-type (+/+), heterozygous (+/ ic ), and homozygous ( ic / ic ) granulocytic forms of EPRO cells were analyzed for the expression of multiple lamins and inner nuclear envelope proteins by immunostaining and immunoblotting techniques. The heterochromatin architecture was also examined by immunostaining for histone lysine methylation. Results Wild-type (+/+) and heterozygous (+/ ic ) granulocytic forms revealed ring-shaped nuclei and contained LBR within the nuclear envelope; ic / ic granulocytes exhibited smaller ovoid nuclei devoid of LBR. The pericentric heterochromatin of undifferentiated and granulocytic ic / ic cells was condensed into larger spots and shifted away from the nuclear envelope, compared to +/+ and +/ ic cell forms. Lamin A/C, which is normally not present in mature granulocytes, was significantly elevated in LBR-deficient EPRO cells. Conclusions Our observations suggest roles for LBR during granulopoiesis, which can involve augmenting nuclear membrane growth, facilitating compartmentalization of heterochromatin, and promoting downregulation of lamin A/C expression.

The major human blood granulocyte, the neutrophil, is an essential component of the innate immunity system, emigrating from blood vessels and migrating through tight tissue spaces to the site of bacterial or fungal infection where they kill and phagocytose invading microbes. Since the late nineteenth century, it has been recognized that the human neutrophil nucleus is distinctly not ovoid as in other cell types, but possesses a lobulated (segmented) shape. This deformable nucleus enhances rapid migration. Recent studies have demonstrated that lamin B receptor (LBR) is necessary for the non-ovoid shape. LBR is an integral membrane protein of the nuclear envelope. A single dominant mutation in humans leads to neutrophils with hypolobulated nuclei (Pelger-Huet anomaly); homozygosity leads to ovoid granulocyte nuclei. Interestingly, LBR is also an enzyme involved in cholesterol metabolism. Homozygosity for null mutations is frequently lethal and associated with severe skeletal deformities. In addition to the necessity for LBR, formation of the mature granulocyte nucleus also depends upon lamin composition and microtubule integrity. These observations are part of a larger question on the relationships between nuclear shape and cellular function.

Mature blood neutrophils (polymorphonuclear granulocytes) have characteristically complex nuclear shapes. The human neutrophil nucleus generally possesses 3-4 lobes; the mouse neutrophil nucleus frequently resembles a twisted toroid with a central hole. Myeloid tissue culture systems (e.g., human HL-60 and murine MPRO) can be induced to differentiate in vitro towards neutrophils by addition of retinoic acid, exhibiting the characteristic nuclear shape changes. Confocal immunostaining and thin-section transmission electron microscopic image data from differentiated HL-60 and MPRO cells clearly demonstrate proximity of the centrosomal region (containing dynein, gamma-tubulin and C-Nap1) to regions of granulocytic nuclear indentations. In addition, the centrosomal region, flanked by the Golgi apparatus, is shown to be present within the central hole of the toroidal mouse granulocyte nucleus. A role for the centrosomal region and associated microtubules in molding granulocytic nuclear shape is suggested.

Thirty years ago, our conception of chromatin structure underwent a total metamorphosis as the nucleosome era began. In Kurosawa's classic movie 'Rashomon' (1951), each participant had a different perspective of the same pivotal event. This review outlines our perception of history.

Epichromatin, the surface of chromatin facing the nuclear envelope in an interphase nucleus, reveals a “rim” staining pattern with specific mouse monoclonal antibodies against histone H2A/H2B/DNA and phosphatidylserine epitopes. Employing a modified ChIP-Seq procedure on undifferentiated and differentiated human leukemic (HL-60/S4) cells, >95% of assembled epichromatin regions overlapped with Alu retrotransposons. They also exhibited enrichment of the AluS subfamily and of Alu oligomers. Furthermore, mapping epichromatin regions to the human chromosomes revealed highly similar localization patterns in the various cell states and with the different antibodies. Comparisons with available epigenetic databases suggested that epichromatin is neither “classical” heterochromatin nor highly expressing genes, implying another function at the surface of interphase chromatin. A modified chromatin immunoprecipitation procedure (xxChIP) was developed because the studied antibodies react generally with mononucleosomes and lysed chromatin. A second fixation is necessary to securely attach the antibodies to the epichromatin epitopes of the intact nucleus.

Ultrastructural studies conducted in situ using conventional transmission electron microscopy have had relatively little impact on defining the structural organization of chromatin. This is due to the fact that in routine transmission electron microscopy, together with the deoxyribonucleoprotein, many different intermingled substances are contrasted, masking the ultrastructure of chromatin. By selective staining of DNA in thin sections, using the Feulgen-like osmium-ammine reaction, these drawbacks have been overcome and worthwhile data have been obtained both on the gross morphology and the ultrastructural-functional organization of chromatin in situ. In the present study these results are reviewed and discussed in light of recent achievements in both interphase nuclear chromatin compartmentalization in interphase nuclei and in the structural organization of chromatin fibers in transcriptionally active and inactive chromatin.

Retinoic acid (RA) treatment of HL-60 cells in vitro induces granulocytic differentiation, involving reorganization of the nucleus and cytoplasm, development of chemoattractant-directed migration, and eventual apoptosis. The present studies with HL-60/S4 cells document that major elements of the cytoskeleton are changed: actin increases by 50%; vimentin decreases by more than 95%. The cellular content of α-tubulin does not significantly change; but the centrosomal–microtubule (MT) array moves away from the lobulating nucleus. Cytoskeletal-modifying chemicals modulate this polarized reorganization: Taxol and cytochalasin D enhance centrosome movement; nocodazole reverses it. Cytoskeletal-modifying chemicals do not appear to affect nuclear lobulation or the integrity of envelope-limited chromatin sheets (ELCS). Employing bcl-2-overexpressing HL-60 cells permitted demonstration of nuclear lobulation, ELCS formation, and centrosome–MT movement concomitantly during RA-induced differentiation, implying independence between the cellular reorganization and apoptotic programs. RA appears to promote an inherent potential in HL-60 cells for cytoskeletal polarization, likely to be important for chemoattractant-directed cell migration, an established characteristic of mature granulocytes.

Molecular features of imprinted genes include differences in expression, methylation, and the timing of DNA replication between parental alleles. Whereas methylation differences always seem to be associated with differences in expression, differences in the timing of replication between parental homologs are not always seen at imprinted loci. These observations raise the possibility that differences in replication timing may not be an essential feature underlying genomic imprinting. In this study, we examined the timing of replication of the two alleles of the imprinted RSVIgmyc transgene in individual embryonic cells using fluorescence in situ hybridization (FISH). The cis-acting signals for RSVIgmyc imprinting are within RSVIgmyc itself. Thus, allele-specific differences in replication, if they indeed govern RSVIgmyc imprinting, should be found in RSVIgmyc sequences. We found that the parental alleles of RSVIgmyc, which exhibit differences in methylation, replicated at the same time. Synchronous replication was also seen in embryonic cells containing a modified version of RSVIgmyc that exhibited parental allele differences in both methylation and expression. These findings indicate that maintenance of expression and methylation differences between alleles does not require a difference in replication timing. The differences in replication timing of endogenous imprinted alleles detected by FISH might therefore reflect structural differences between the two alleles that could be a consequence of imprinting or, alternatively, could be unrelated to imprinting.

Neutrophils are characterized by their distinct nuclear shape, which is thought to facilitate the transit of these cells through pore spaces less than one-fifth of their diameter. We used human promyelocytic leukemia (HL-60) cells as a model system to investigate the effect of nuclear shape in whole cell deformability. We probed neutrophil-differentiated HL-60 cells lacking expression of lamin B receptor, which fail to develop lobulated nuclei during granulopoiesis and present an in vitro model for Pelger-Huet anomaly; despite the circular morphology of their nuclei, the cells passed through micron-scale constrictions on similar timescales as scrambled controls. We then investigated the unique nuclear envelope composition of neutrophil-differentiated HL-60 cells, which may also impact their deformability; although lamin A is typically down-regulated during granulopoiesis, we genetically modified HL-60 cells to generate a subpopulation of cells with well defined levels of ectopic lamin A. The lamin A-overexpressing neutrophil-type cells showed similar functional characteristics as the mock controls, but they had an impaired ability to pass through micron-scale constrictions. Our results suggest that levels of lamin A have a marked effect on the ability of neutrophils to passage through micron-scale constrictions, whereas the unusual multilobed shape of the neutrophil nucleus is less essential.

Ultra-thin sections of Chironomus salivary glands were stained in a non-Feulgen procedure with osmium ammine-B and imaged at several electron energy-loss windows. For two types of RNP-containing structures (ie Balbiani ring granules and endoplasmic reticulum), a significant spatial correlation was observed between stain distribution and net phosphorus distribution. Non-Feulgen osmium ammine-B staining does not require the use of ultra-thin sections and can approximate the distribution of nucleic acid phosphorus.

The replication band is a large, migrating, macronuclear domain that is the site of DNA synthesis in hypotrichous ciliated protozoa. A number of agents that produce inactivation of this structure and its replicational activity are described here. These agents include heat shock, aphidicolin, cell crowding, various cAMP phosphodiesterase inhibitors and a calmodulin inhibitor. With the exception of aphidicolin, which has a direct inhibitory effect upon DNA polymerases, the mechanisms of inactivation are presently unknown. The inactivating properties of cAMP phosphodiesterase inhibitors suggest that intracellular cAMP levels may influence replication band structure and function. Supplementary key words. Autoradiography, electron microscopy, in situ hybridization. HE sole site of DNA synthesis in the macronuclei (MAC) T of hypotrichous ciliated protozoa is the replication band (RB) 115, 20, 39, 401. It has been most studied in the MAC of the fresh water ciliate Euplotes eurystomus. The single MAC is elongate and C-shaped; i.e. ca. 100-150 pm long by 10-15 pm wide. The single MIC is the "germline" micronucleus, ca. 3 pm diameter, with an independent S phase immediately following cytokinesis. During MAC S phase, two RB appear simulta- neously at the tips of a MAC. Both RB migrate to the midpoint of the elongate MAC as S phase progresses for about 10-12 hr. The two RB fuse at the termination of S, the MAC shortens and becomes pinched in half during cytokinesis. During the entire S phase the RB maintain a unique ultrastructural strati- fication (30, 3 1, 401. The migrating RE is ca. 5 pm in length and composed of two structural zones: a forward zone (FZ) consisting of a mesh of 40-50-nm chromatin fibers; and a pale- staining rear zone (RZ) containing fine ca. 10-nm fibers. In front of and behind the RB, chromatin is organized into condensed granules. Autoradiographic EM studies have demonstrated that the site of DNA synthesis is the junction between FZ and RZ (25) (Stevens, A. R. 1963, Electron microscope autoradiog- raphy of DNA and RNA synthesis in Euplotes eurystomus. J. Cell Biol., 19:67a). Replication bands exhibit distinctive cyto- chemical and immunochemical properties ( 1-3, 30, 33, 34, 361. A clue towards understanding the structural integrity of RB may be derived from the recent observation of non-chromatin 10- nm filaments within Euplotes MAC in condensed chromatin granules (CG) and in the FZ (29). Conceivably these 10-nm filaments might function to organize the chromatin molecules into the various nuclear compartments and functional states. Organization of MAC chromatin is an obvious necessity when it is recalled that each MAC contains ca. lo8 gene-size linear DNA fragments of average size ca. 2 kb (21, 221. Each type of MAC DNA fragment appears to possess a single coding region flanked by non-transcribed regions and terminated by identical telomeric nucleotide sequences. Otherwise, the MAC DNA mol- ecules exist as reasonably normal chromatin molecules with inner histones, histone H1 and nucleosomes (8, 9, 171.

Nuclear envelope-limited chromatin sheets (ELCS) form during excessive interphase nuclear envelope growth in a variety of cells. ELCS appear as extended sheets within the cytoplasm connecting distant nuclear lobes. Cross-section stained images of ELCS, viewed by transmission electron microscopy, resemble a sandwich of apposed nuclear envelopes separated by ∼30 nm, containing a layer of parallel chromatin fibers. In this study, the ultrastructure of ELCS was compared by three different methods: (1) aldehyde fixation/dehydration/plastic embedding/sectioning and staining, (2) high-pressure freezing/freeze substitution into plastic/sectioning and staining, and (3) high-pressure freezing/cryo-sectioning/cryo-electron microscopy. ELCS could be clearly visualized by all three methods and, consequently, must exist in vivo and are not fixation artifacts. The ∼30-nm chromatin fibers could only be observed following aldehyde fixation; none were seen in cryo-sections. Electron microscopic tomography tangential views of aldehyde-fixed ELCS suggested an ordering of the separate chromatin fibers adjacent to the nuclear envelope. Possible mechanisms of this chromatin ordering are discussed.

The principal human blood granulocyte (neutrophil) possesses a lobulated and deformable nucleus, important to facilitate rapid egress from blood vessels as these cells migrate to sites of bacterial or fungal infection. This unusual nuclear shape is a product of elevated levels of an integral membrane protein of the nuclear envelope lamin B receptor (LBR) and of decreased amounts of lamin A/C. In humans, a genetic deficiency of LBR produces Pelger-Huet anomaly, resulting in blood neutrophils that exhibit hypolobulated nuclei with redistributed heterochromatin. Structural changes in nuclear architecture occur during granulopoiesis within bone marrow. The exact mechanisms of this nuclear shape change and of heterochromatin redistribution remain largely unknown. As a tool to facilitate analysis of these mechanisms, a stable LBR knockdown subline of HL-60 cells was established. During in vitro granulopoiesis induced with retinoic acid, the LBR knockdown cells retain an ovoid shaped nucleus with reduced levels of lamin A/C; while, the parent cells develop highly lobulated nuclei. In contrast, macrophage forms induced in LBR knockdown cells by in vitro treatment with phorbol ester were indistinguishable from the parent cells, judged by both nuclear shape and attached cell morphology. The capability of differentiation of LBR knockdown HL-60 cells should facilitate a detailed analysis of the molecular relationship between LBR levels, granulocyte nuclear shape and heterochromatin distribution.

The Balbiani Rings (BR) in the polytene chromosomes of Chironomus salivary glands are intense sites of transcription. The nascent RNPs fold during transcription into 40-50-nm granules, containing in the mature transcript approximately 37-kb RNA. Using a new nucleic acid specific stain, osmium ammine B on Lowicryl sections, in combination with electron energy filtered imaging of sections containing BR granules, we demonstrate a RNA-rich particulate substructure (10-nm particle diameter; 10-12 particles per BR granule). Elemental imaging supports that these particles are enriched in phosphorus. The possible relationship of these RNA-rich particles to ribonucleosomes is discussed, as well as models for their arrangement in the mature BR granules.

Lamin B receptor (LBR) is an integral membrane protein of the interphase nuclear envelope (NE). The N-terminal end resides in the nucleoplasm, binding to lamin B and heterochromatin, with the interactions disrupted during mitosis. The C-terminal end resides within the inner nuclear membrane, retreating with the ER away from condensing chromosomes during mitotic NE breakdown. Some of these properties are interpretable in terms of our current structural knowledge of LBR, but many of the structural features remain unknown. LBR apparently has an evolutionary history which brought together at least two ancient conserved structural domains (i.e., Tudor and sterol reductase). This convergence may have occurred with the emergence of the chordates and echinoderms. It is not clear what survival values have maintained LBR structure during evolution. But it seems likely that roles in post-mitotic nuclear reformation, interphase NE growth and compartmentalization of nuclear architecture might have provided some evolutionary advantage to preservation of the LBR gene. REVIEW

The major blood granulocyte (neutrophil) is rapidly recruited to sites of bacterial and fungal infections. It is a highly malleable cell, allowing it to squeeze out of blood vessels and migrate through tight tissue spaces. The human granulocyte nucleus is lobulated and exhibits a paucity of nuclear lamins, increasing its capability for deformation. The present study examined the existence of protein connections between the nuclear envelope and cytoskeletal elements (the LINC complex) in differentiated cell states (i.e. granulocytic, monocytic and macrophage) of the human leukemic cell line HL-60, as well as in human blood leukocytes. HL-60 granulocytes exhibited a deficiency of several LINC complex proteins (i.e. nesprin 1 giant, nesprin 2 giant, SUN1, plectin and vimentin); whereas, the macrophage state revealed nesprin 1 giant, plectin and vimentin. Both states possessed SUN2 in the nuclear envelope. Parallel differences were observed with some of the LINC complex proteins in isolated human blood leukocytes, including macrophage cells derived from blood monocytes. The present study documenting the paucity of LINC complex proteins in granulocytic forms, in combination with previous data on granulocyte nuclear shape and nuclear envelope composition, suggest the hypothesis that these adaptations evolved to facilitate granulocyte cellular malleability.

Objective. The capacity of neutrophils to eradicate bacterial infections is dependent on normal development and activation of functional responses, which include chemotaxis and generation of oxygen radicals during the respiratory burst. A unique feature of the neutrophil is its highly lobulated nucleus, which is thought to facilitate chemotaxis, but may also play a role in other critical neutrophil functions. Nuclear lobulation is dependent on expression of the inner nuclear envelope protein, the lamin B receptor (LBR), mutations of which cause hypolobulated neutrophil nuclei in human Pelger-Huet anomaly and the ''ichthyosis'' (ic) phe- notype in mice. In this study, we have investigated roles for LBR in mediating neutrophil development and activation of multiple neutrophil functions, including chemotaxis and the respiratory burst. Materials and Methods. A progenitor EML cell line was generated from an ic/ic mouse, and derived cells that lacked LBR expression were induced to mature neutrophils and then exam- ined for abnormal morphology and functional responses. Results. Neutrophils derived from EML-ic/ic cells exhibited nuclear hypolobulation identical to that observed in ichthyosis mice. The ic/ic neutrophils also displayed abnormal chemotaxis, supporting the notion that nuclear segmentation augments neutrophil extravasation. Further- more, promyelocytic forms of ic/ic cells displayed decreased proliferative responses and pro- duced a deficient respiratory burst upon terminal maturation. Conclusions. Our studies of promyelocytes that lack LBR expression have identified roles for LBR in regulating not only the morphologic maturation of the neutrophil nucleus, but also proliferative and functional responses that are critical to innate immunity. 2008 ISEH

The human blood granulocyte (neutrophil) is adapted to find and destroy infectious agents. The nucleus of the human neutrophil has a segmented appearance, consisting of a linear or branched array of three or four lobes. Adequate levels of lamin B receptor (LBR) are necessary for differentiation of the lobulated nucleus. The levels of other components of the nuclear envelope may also be important for nuclear shape determination. In the present study, immunostaining and immunoblotting procedures explored the levels of various components of the nuclear envelope and heterochromatin, comparing freshly isolated human neutrophils with granulocytic forms of HL-60 cells, a tissue culture model system. In comparison to granulocytic HL-60 cells, blood neutrophil nuclear envelopes contain low-to-negligible amounts of LBR, lamins A/C, B1 and B2, LAP2beta and emerin. Surprisingly, a "mitotic" chromosome marker, H3(S10)phos, is elevated in neutrophil nuclei, compared to granulocytic HL-60 cells. Furthermore, neutrophil nuclei appear to be more fragile to methanol fixation, than observed with granulocytic HL-60 cells. Thus, the human neutrophil nucleus appears to be highly specialized, possessing a paucity of nuclear envelope-stabilizing proteins. In consequence, the neutrophil nucleus appears to be very malleable, supporting rapid migration through tight tissue spaces.

A combinatorial mutagenesis strategy was used to create a collection of nearly 500 variants of human interleukin 3 (IL-3), each with four to nine amino acid substitutions clustered within four linear, nonoverlapping regions of the polypeptide. The variants were secreted into the periplasm of Escherichia coli and supernatants were assayed for IL-3 receptor-dependent cell proliferation activity. Sixteen percent of the variants, containing "region-restricted" substitutions, retained substantial proliferative activity through two rounds of screening. A subset of these was combined to yield variants with substitutions distributed through approximately half of the polypeptide. With one exception, "half-substituted" variants exhibited proliferative activity within 3.5-fold of native IL-3. A subset of the "half-substituted" variants was combined to yield "fully substituted" IL-3 variants having 27 or more substitutions. The combination of the substitutions resulted in a set of polypeptides, some of which exhibit increased proliferative activity relative to native IL-3. The elevated hematopoietic potency was confirmed in a methylcellulose colony-forming unit assay using freshly isolated human bone marrow cells. A subset of the multiply substituted proteins exhibited only a modest increase in inflammatory mediator (leukotriene C4) release. The molecules also exhibited 40- to 100-fold greater affinity for the alpha subunit of the IL-3 receptor and demonstrated a 10-fold faster association rate with the alpha-receptor subunit. The multiply substituted IL-3 variants described in this study provide a unique collection of molecules from which candidates for clinical evaluation may be defined and selected.

Employing in situ nick translation on isolated macronuclei from the ciliate Euplotes eurystomus, the replication band exhibited heightened DNase I sensitivity. This enhanced reactivity was not significantly affected by decreased replication induced with heat shock or added inhibitors, implying that the underlying chromatin modulations are reasonably stable. The nuclease sensitivity of the replication band is probably related to the ‘maturation’ of nascent chromatin, since transcriptional activity is known to be absent within the band. An additional observation of this study was the in vivo incorporation of halogen-deoxynucleosides into replication bands, facilitating future studies of matrix-associated nascent DNA.

The human leukemic cell line (HL-60) can be induced to differentiate in vitro to granulocytic form with retinoic acid (RA), or to monocytic/macrophage form with phorbol ester (TPA). The granulocytic form acquires nuclear lobulation, nuclear envelope-limited chromatin sheets (ELCS), and cytoskeletal polarization, none of which are acquired following treatment with TPA. Immunoblotting analyses and capillary zone electrophoresis demonstrated that following RA treatment: lamins A/C and B1, and vimentin decreased to negligible amounts; LAP2β, lamin B2 and emerin remained essentially unchanged; lamin B receptor (LBR) increased markedly; histone subtypes H1.4 and 1.5 exhibited dephosphorylation. Following TPA treatment: lamins A/C and B1, B2 and vimentin increased in amount; LAP2β and emerin remained essentially unchanged; LBR increased markedly; histone subtypes H1.4 and 1.5 exhibited dephosphorylation. Emerin, which was cytoplasmic in undifferentiated or granulocytic cells, localized into the nuclear envelope following TPA. Normal human granulocytes revealed compositional differences compared to granulocytic forms of HL-60, namely increased vimentin and appearance of histone subtype H1.3. A working hypothesis for nuclear lobulation postulates a combination of: increased nuclear envelope deformability due to lamins A/C and B1 deficiency; an increase in nuclear surface area/volume; an increase in chromatin–nuclear envelope interactions.

Employing several antibodies to phosphorylated protein epitopes, we demonstrate by immunostaining that the macronuclear replication band (RB) of the ciliated protozoan Euplotes eurystomus contains a high concentration of phosphoproteins. Enrichment is principally within the rear zone of the RB, the region of DNA synthesis and chromatin assembly. By immunoblot analysis, the various antibodies reacted with a diversity of macronuclear phosphoproteins, one of which was phosphorylated histone Hl. This diversity of phosphoproteins was also supported by examination of the macronuclear matrix generated by high NaCl extraction. Available evidence clearly indicates that the ultrastructural wave of chromatin modulation accompanying DNA replication is spatially correlated with a wave of localized nuclear protein phosphorylation.

In order to study the potential role of cysteinyl residues in catalysis and inhibition of human aldose reductase, mutants containing cysteine to serine substitution at positions 80 (ALR2:C80S), 298 (ALR2:C298S), and 303 (ALR2:C303S) were constructed. Mutation of Cys298 resulted in the most profound changes, as ALR2:C298S displayed 4- to 5-fold elevation in K'm(NADPH), K'm(DL-glyceraldehyde), and kcat(DL-glyceraldehyde) relative to wild type aldose reductase as well as a 10-fold higher Ki for the aldose reductase inhibitor sorbinil. Wild type and mutant reductases were equally sensitive to tolrestat, a structurally different reductase inhibitor. Carboxymethylation of the wild type enzyme or the C80S and C303S mutants led to a modest decrease in kcat as well as an increase in K'm(DL-glyceraldehyde) and Ki(sorbinil). These parameters were not significantly changed when ALR2:C298S was subjected to carboxymethylation. Lithium sulfate caused activation of ALR2:WT, C80S, and C303S but did not significantly affect the activity of ALR2:C298S. The differential sensitivity of wild type and mutant reductases to inhibition by sorbinil and tolrestat, before and after carboxymethylation, indicates that these inhibitors bind at different sites. These results suggest that Cys-298 is present near the active site and constitutes a regulatory group which controls the catalytic activity and inhibitor sensitivity of the enzyme.

The effect of overexpression of the heat shock chaperone genes dnaK and groESL on heterologous protein production in Escherichia coli was examined, using a set of related human procollagenase proteins. A diverse range of effects on protein solubility, secretion, and accumulation was observed, and these effects were highly dependent on the particular chaperone/procollagenase pairing involved. Both chaperones caused a large increase in the apparent solubility of a fusion of the LamB signal peptide to procollagenase. GroESL had no effect on the accumulation of mature (secreted) procollagenase, while DnaK suppressed secretion considerably. In the absence of a signal peptide, overexpression of either chaperone resulted in a dramatic increase in both solubility and accumulation of procollagenase. The 10-fold increase in accumulation was associated with an increase in in vivo protein half-life.

Exposure of the human leukemic cell line (HL-60) to 1 microM retinoic acid (RA) induces in vitro granulopoiesis, including the development of lobulated nuclei. Ultrastructural studies, presented here, demonstrate the formation of extensive quantities of nuclear envelope-limited chromatin sheets (ELCS), in addition to nuclear lobulation, following treatment with RA. ELCS contain DNA, as shown by the Feulgen-like electron microscope stain osmium ammine-B. Lamin B was demonstrated in ELCS by immunoelectron microscopy with colloidal gold-labeled antibody. Formation of ELCS occurred in Bcl2-overexpressing HL-60 cell sublines with suppressed apoptotic cell death, indicating separable mechanisms for ELCS formation and apoptosis. Immunofluorescent and immunoblotting procedures demonstrated modulations in the amounts and distribution of nuclear envelope-associated components. Total amounts of lamins A/C and cytoplasmic vimentin were reduced by RA treatment. The amounts of lamin B, lamin B receptor (LBR), and lamina-associated polypeptide 2 (LAP2) did not exhibit significant quantitative changes, but acquired heterogeneous staining patterns on the nuclear envelope. RA induced the appearance of low-molecular-weight LBR-related proteins. This study demonstrated the parallel induction of lobulated nuclei and of ELCS and the modulation of nuclear envelope components following exposure of HL-60 to retinoic acid.

We have examined the patterns of compartmentalization of several mammalian proteins in Escherichia coli which do not have signal peptides or functional signal peptide equivalents. These proteins include (i) human proapolipoprotein A-I (proapoA-I), a 249-residue protein which contains a hexapeptide NH2-terminal prosegment plus a mature domain of 243 residues comprised of tandemly arrayed, docosapeptide repeats with predicted amphipathic alpha-helical structure; (ii) the mature apoA-I molecule without its prosegment; (iii) mouse interleukin-1 beta (IL-1 beta), a 17-kDa protein which is composed of 12 beta strands that form a tetrahedral structure; and (iv) the 31-kDa precursor of IL-1 beta, proIL-1 beta. Efficient expression of these proteins in E. coli was achieved using a plasmid that contains the nalidixic acid-inducible recA promoter and ribosome binding site from the gene 10 leader of bacteriophage T7. In induced cultures the mammalian proteins represented up to 20% of the total bacterial protein mass. Surprisingly, cell fractionation using cold (osmotic) shock indicated that proapoA-I, apoA-I, and IL-1 beta, but not its 31-kDa precursor, were segregated into the periplasmic space with high efficiency: the ratio of periplasmic space/spheroplast distribution ranged from 0.6 to 1.1 in cells harvested 60-180 min after nalidixic acid induction. Not only was this compartmentalization efficient but it was also selective: analysis of the osmotic shock fractions revealed that the periplasmic space preparations were not contaminated with cytoplasmic proteins (e.g. phosphoglycerate dehydrogenase). Sequential Edman degradation showed that these proteins had not undergone any NH2-terminal proteolytic processing. The mammalian proteins did not affect the export of a prototypic bacterial preprotein, beta-lactamase. Together the data suggest that osmotic shock fractionation of E. coli may facilitate the purification of functional foreign proteins produced in this prokaryote. They also raise the possibility that structural elements in these proteins other than conventional signal peptides may effect periplasmic targeting in E. coli.

Neutrophils and macrophages are professional phagocytes that are essential to the immune response and that have developmental and functional similarities. They derive from a common myeloid progenitor and require common transcription factors for their differentiation, such as PU.1 and the CCAAT/enhancer binding proteins C/EBPα and C/EBPε. However, the morphologic maturation of these two lineages is distinct; neutrophil progenitors undergo a progressive series of nuclear changes that culminate in the formation of lobulated nuclei, whereas macrophage progenitors first form circulating monocytes that contain indented nuclei, and then form mature tissue macrophages that contain small, spherical nuclei. To date, little is known regarding the mechanism that regulates nuclear morphogenesis of either lineage. In recent efforts to further understand neutrophil differentiation, we and others have demonstrated that nuclear lobulation in both human and mouse neutrophils is dependent on lamin B receptor (LBR) expression. LBR is an integral membrane protein of the inner nuclear envelope that interacts with B-type lamins and heterochromatin. We have shown that LBR expression is significantly upregulated during all-trans retinoic acid (ATRA)-induced neutrophil differentiation of murine hematopoietic progenitor EML/EPRO cells, which parallels changes in LBR expression exhibited by ATRA-induced human leukemic HL-60 cells. Both models show decreased expression of lamin A/C during neutrophil maturation, whereas lamin B expression remains constant. Importantly, loss of LBR protein expression causes hypolobulation of neutrophil nuclei that is characteristic of the Pelger-Huët anomaly in both mice and humans. These studies support the notion that dynamic changes in the expression of LBR and perhaps lamin A/C play critical roles in neutrophil nuclear maturation. Studies that examine macrophage nuclear maturation, however, have been limited to those performed on HL-60 cells, which showed that LBR expression increased during phorbol ester (TPA) induction. We have now examined the expression of LBR in two additional stem cell factor (SCF)-dependent murine myeloid cell lines that can be induced toward mature neutrophils, pBIM and SCF ER-Hoxb8, as well as in a GM-CSF-dependent ER-Hoxb8 cell line that differentiates into mature macrophages. The pBIM cell line was generated by insertional mutagenesis of mouse marrow cells with the empty pBabe-puro vector. The ER-Hoxb8 cell lines were generated from mouse marrow that was transduced with an estrogen-regulated Hoxb8, which arrests myeloid differentiation. Similar to our findings in EML/EPRO cells, Lbr transcription was upregulated in pBIM cells upon G-CSF-induced neutrophil differentiation, and LBR protein levels significantly increased in SCF ER-Hoxb8 cells upon estrogen withdrawal. By comparison, Lbr transcription dramatically increased during the initial stages of macrophage differentiation of GM-CSF ER-Hoxb8 progenitors, but then decreased in mature macrophages. Initial increases in Lbr transcription during both neutrophil and macrophage differentiation paralleled upregulated expression of both PU.1 and C/EBPε. We also detected increased expression of Lbr in pBIM cells that were generated from a C/EBPε-knockout mouse. This result was surprising given that very recent studies have demonstrated that C/EBPε can directly activate the Lbr promoter. Finally, lamin A/C expression increased during macrophage differentiation of GM-SCF ER-Hoxb8 cells, but was minimally detected in uninduced or differentiated SCF ER-Hoxb8 cells. Together our data indicate that changes in the expression of LBR and lamin A/C play important roles during the maturation of both granulocyte and macrophage lineages, and that PU.1 and/or C/EBPε drive these changes in expression. However, our analyses of C/EBPε-knockout cells indicate that LBR transcription is not absolutely dependent on C/EBPε expression. Current studies to identify the expression and interplay of nuclear envelope proteins during neutrophil and macrophage differentiation that employ these cell line models as well as primary marrow progenitors will further define the mechanisms that regulate morphologic maturation of two important phagocytes.

The tightly centromere-linked gene SPO15 is essential for meiotic cell division in the yeast Saccharomyces cerevisiae. Diploid cells without the intact SPO15 gene product are able to complete premeiotic DNA synthesis and genetic recombination, but are unable to traverse the division cycles. Electron microscopy of blocked cells reveals a duplicated but unseparated spindle-pole body. Thus cells are unable to form a bipolar spindle. Sequence analysis of SPO15 DNA reveals an open reading frame that predicts a protein of 704 amino acids. This protein is identical to VPS1, a gene involved in vacuolar protein sorting in yeast which has significant sequence homology (45% overall, 66% over 300 amino acids) to the microtubule bundling-protein, dynamin. The SPO15 gene product expressed in Escherichia coli can be affinity-purified with microtubules. SPO15 encodes a protein that is likely to be involved in a microtubule-dependent process required for the timely separation of spindle-pole bodies in meiosis.

During granulopoiesis in the bone marrow, the nucleus differentiates from ovoid to lobulated shape. Addition of retinoic acid (RA) to leukemic HL-60 cells induces development of lobulated nuclei, furnishing a convenient model system for nuclear differentiation during granulopoiesis. Previous studies from our laboratory have implicated nuclear envelope composition as playing important roles in nuclear shape changes. Specifically noted were: 1) a paucity of lamins A/C and B1 in the undifferentiated and RA treated cell forms; 2) an elevation of lamin B receptor (LBR) during induced granulopoiesis. The present study demonstrates that perturbation of cytoskeletal elements influences nuclear differentiation of HL-60 cells. Because of cytotoxicity from prolonged exposure to cytoskeleton-modifying drugs, most studies were performed with a Bcl-2 overexpressing HL-60 subline. We have found that: 1) nocodazole prevents RA induction of lobulation; 2) taxol induces lobulation and micronuclear formation, even in the absence of RA; 3) cytochalasin D does not inhibit RA induced nuclear lobulation, and prolonged exposure induces nuclear shape changes in the absence of RA. The present results, in the context of earlier data and models, suggest a mechanism for granulocytic nuclear lobulation. Our current hypothesis is that the nuclear shape change involves factors that increase the flexibility of the nuclear envelope (reduced lamin content), augment connections to the underlying heterochromatin (increased levels of LBR) and promote distortions imposed by the cytoskeleton (microtubule motors creating tension in the nuclear envelope).

Tissue factor pathway inhibitor (TFPI) is a potent inhibitor of the blood coagulation factor VIIa-tissue factor complex, as well as a direct inhibitor of factor Xa. Intravenously administered TFPI is rapidly cleared from circulation predominantly via liver. We previously reported that the low density lipoprotein receptor-related protein (LRP), a multifunctional endocytic receptor, mediates the uptake and degradation of TFPI in hepatoma cells. This process is inhibited by a 39-kDa receptor-associated protein which binds to LRP and regulates its ligand binding activity. However, a distinct, low affinity binding site (perhaps heparin sulfate proteoglycans, HSPGs) on the endothelium and liver is thought to be responsible for the majority of TFPI cell surface binding. In the current study, we investigated the role of LRP and this second binding site in the clearance of 125I-TFPI in vivo using competitors and inhibitors of the receptors. Mice overexpressing the 39-kDa protein via adenoviral-mediated gene transfer displayed diminished plasma clearance of 125I-TFPI. Blockade of cell surface HSPGs sites by incubation with the positively charged molecule, protamine, inhibited 125I-TFPI binding to the hepatoma cells in vitro. In addition, preadministration of protamine in vivo prolonged the plasma clearance of 125I-TFPI in a dose-dependent manner. However, a dramatic increase of the plasma half-life of 125I-TFPI and virtual elimination of 125I-TFPI clearance was observed in mice overexpressing the 39-kDa protein and administered protamine. Taken together, our results suggest that two receptor mechanisms are involved in the clearance of TFPI in vivo.

A deletion variant of human interleukin-3, hIL-3(15-125), was produced in the periplasmic space of Escherichia coli and had full activity in an AML193.1.3 cell proliferation assay. Libraries of random single-amino acid substitutions were constructed at each of 105 positions in the gene for hIL-3(15-125). Approximately eight single-site substitutions at each position were produced in osmotic shock fractions and screened for activity. 15 mutants were found with bioactivity of 5-26-fold greater than that of native hIL-3. The majority of amino acids in hIL-3(15-125) could be substituted without substantial loss of activity. Substitution of residues predicted to be in the hydrophobic core of the protein often resulted in reduced activity and/or low accumulation levels. Only five residues predicted to be on the surface of the protein were intolerant of substitution and hence are candidates for sites of interaction with the receptor. We therefore propose that the majority of residues in hIL-3 serve a structural role and permit the display of a few key residues in the correct configuration for recognition by the receptor.

Mouse Interleukin 4 is a 20-kDa glycoprotein, synthesized by activated T lymphocytes and mast cells, which regulates the growth and/or differentiation of a broad spectrum of target cells of the immune system, including B and T lymphocytes, macrophages, and hematopoietic progenitor cells. Using an inducible recA promoter and the g10-L ribosome-binding site, recombinant non-glycosylated interleukin 4 (IL-4) was expressed as 17% of total cellular protein in Escherichia coli inclusion bodies, as a reduced, inactive 14.5-kDa polypeptide. The protein was refolded and aggregates dissociated when three disulfide bonds were reformed by slowly decreasing the concentration of guanidine hydrochloride and cysteine. The oxidized monomer was purified to homogeneity by sequential ion-exchange and size exclusion chromatography. When compared with native IL-4, E. coli-derived IL-4 displayed an identical specific activity of 4-7 x 10(7) units/mg. This recombinant IL-4 contained a three-amino-acid NH2-terminal extension, which did not affect its biological activity. Purified biologically active protein consisted of three isoforms as shown by two-dimensional gel electrophoresis, with a pI greater than 9.0. These data suggest that neither glycosylation nor the NH2 terminus of mouse IL-4 play a critical role in contributing to its in vitro biological activity.

Euplotes is a hypotrichous ciliate that contains both a macronucleus (MAC) and a micronucleus in the same cell. The DNA in the MAC is composed of small, linear highly amplified fragments, each of which contains one gene, two telomeres and all the necessary regulatory elements. Although there is a large amount of condensed chromatin in the MAC, it appears not to be organized in 30 nm fibers as seen in higher eukaryotes (9). Since histone HI is responsible for the formation of the 30 nm fiber, analysis of this protein in Euplotes may provide some insight into the unique chromatin structure in the MAC. Histone HI from the Euplotes MAC has been previously

Detergent permeabilized Euplotes eurystomus (a fresh water hypotrichous ciliate) was reacted with monoclonal and polyclonal antibodies specific for either detyrosinated or tyrosinated α-tubulin (Glu- or Tyr-tubulin). The isolated cytoskeleton-nuclear complex was examined by Western immunoblotting and by immunofluorescent and electron microscopic methods. Both Glu- and Tyr-tubulins were detected by immunoblot analysis. Immunofluorescent microscopy indicated that the α-tubulin isotypes are concentrated in different regions of permeabilized cells: Glu-tubulin is located primarily in cirri, membranelles, and surrounding the macro- and micronuclei. Tyr-tubulin is principally at the bases of cirri and membranelles. This differential distribution of α-tubulin isotypes is discussed in terms of current concepts concerning the correlation of tubulin post-translational modifications to microtubule stability. Confocal immunofluorescent imaging was of critical importance in clearly differentiating the Glu-tubulin isotype surrounding the macro- and micronuclei from a brilliantly fluorescent environment originating from cytoskeletal structures. In conjunction with conventional and stereo-electron microscopy, confocal optical microscopy provided convincing evidence for a “basket” of microtubules surrounding both nuclei.